In scientific research studies in the general area of cell biology, investigations of both soft and hard (mineralized) tissues are commonplace. Examination and analysis of hard tissues such as bones and teeth, the shells of mollusks and other examples of vertebrate, invertebrate, plant, bacterial and other organisms often encounter difficulties since these tissues in many instances must be specially treated to remove their constituent mineral. For investigations to determine nucleic acids, genes, and proteins of both soft and hard tissue cells, a frequent method of processing involves cutting the cells into very thin sections. Again, the demineralization of the hard tissues is required for proper and consistent sectioning.
Decalcification can be accomplished by utilizing any of several different methodologies, including application of chelating reagents, acids or even microwave radiation. There are many products currently on the market and available for demineralization, intended to assist in hard tissue treatment for routine clinical and basic science research in the field of protein analysis.
For aspects of cell biology concerning nucleic acid, and particularly ribonucleic acid (RNA), research and gene studies, additional techniques must be carefully considered. Because gene expression can change within seconds of cell processing, tissues must be handled in a way that does not change or degrade (destroy) the cellular RNA. In the cases of human, animal or plant tissues, RNA analysis had generally required that the samples be snap-frozen in liquid nitrogen at the immediate point of their retrieval upon death or surgical removal. Snap-frozen RNA has proven to be of the highest quality and other methods are usually compared to this standard.
More recently, however, RNA preservation solutions like RNAlater® Solution have been developed to stabilize RNA in animal, human or other tissues without the need for snap freezing. It has been found that, samples stored in RNAlater® Solution were of a quality comparable to samples processed and stored following snap-freezing. This was a significant advance in nucleic acid preservation, since it was often impossible, especially away from the laboratory and out in the field, to have access to or carry the liquid nitrogen necessary for snap freezing.
The importance of RNA preservation has continued to grow with the development of more and more sophisticated genetic analyses using various types of RNA. In particular, researchers and medical professionals have been utilizing gene expression analysis to both diagnose and treat various maladies and for basic scientific research. While each cell in an organism will contain the same genomic DNA and accordingly the same “genes,” only a small fraction of the genes in any particular cell or cell type is ever used at any one time. When a particular gene is activated, the genetic information necessary to create the prescribed protein is transcribed to the ribonucleic acid (RNA) that will be used to make the desired protein.
By identifying and quantifying the RNA in a sample, gene expression analysis makes it possible to determine both what genes are being expressed and, often more importantly, when, where, and in what concentration those genes are being expressed. That is, if the location from which the RNA was recovered can be accurately determined, then the particular cells or cell types that are responsible for the gene expression can likewise be determined. The location of the cells or cell types studied, and, therefore the source of the RNA recovered from the sample, is ordinarily a matter of careful sectioning of the sample to be tested. From the sample section, specific cells, cell types etc. can be selected for analysis using techniques such as manual or laser capture microdissection. Two increasingly common types of gene expression analyses are In-Situ Hybridization (ISH) and laser capture microdissection (LCM).
ISH analysis of RNA requires sections to be cut from chemically fixed tissue samples and then molecular probes are used to label and identify genes of interest in these sections. RNA may be visualized by the labeling in particular areas of the sections and analyzed qualitatively in a temporospatial manner. The detection of labeled genes in hard tissues again requires demineralization to achieve optimal results.
LCM methodology is unique compared to other procedures for gene (and protein) identification in that specific cells of interest, identified by viewing them under a microscope within a population of cells, may be precisely removed from a tissue section using a laser beam. The isolated and so-called captured cells may then be analyzed to address a multitude of questions dealing with gender, disease, drug and other effects on a specimen. RNA obtained following LCM capture of one or several cells in sections may subsequently be assessed quantitatively using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. Resulting data offer insight into biochemical reactions and pathways in a more direct manner than that of ISH. LCM is a microscopic technique requiring tissues to be sectioned onto particular slides of either glass or a thin polymer membrane. Hard tissue study by LCM normally requires demineralization of the sample.
A common approach to analyze specific cell types, individually or as groups of cells spatially, is cryosectioning. Samples are snap-frozen and embedded in a tissue freezing medium available commercially. The frozen tissue or biopsy is sectioned into 4-20 μm slices in a cryostat instrument and kept frozen to preserve the RNA until isolation and analysis. This routine procedure for analysis is easily accomplished on tissues that are by nature soft, i.e., kidney or liver. This approach does make it possible to determine with some precision what cells and cell types are being analyzed.
Analysis of hard tissues like bone, mineralizing cartilage and tendon, dentin, cementum, and enamel, as well as invertebrate shells and other tissues which are too hard to cut or section effectively using conventional means, such as a cryostat has been found, however, to be a major problem. There have developed a variety of approaches to preparing these types of hard tissues for gene expression analysis. One common approach is to snap-freeze and then grind the hard tissue into a powder, and extract the RNA for analysis. While this approach does produce RNA for gene expression analysis relatively quickly without significant degradation, the gene data obtained using this method relate to all of the cells present in the sample. Because of this, it is not possible to determine what specific phenotype or lineage in population of cells/cell types present in the typically mixed cell groupings are producing the RNA obtained.
Another approach is to decalcify the hard tissue sample with acids or chelating agents, thereby softening it so it can be cryosectioned and analyzed. The two most common groups of decalcifying agents known in the art are chelating agents and acids. The acids may be further divided into weak organic (picric, acetic and formic acid) and strong inorganic acids (nitric and hydrochloric acid). The acids dissolve hydroxyapatite mineral with release of calcium ions while chelating agents take up or capture the calcium ions within their structure. The most frequently clinically used chelating agent is ethylenediaminetetraacetic acid (EDTA).
Unfortunately, however, RNA is relatively fragile and tends to break down rapidly after the tissue sample is taken and known decalcification agents have been found to degrade the RNA in the sample. Acids used for decalcification in other procedures are very harsh, and with chelating agents such as EDTA or disodium-EDTA, the decalcification process can take as long as four to six weeks. The RNA recovered using this process is often degraded to the point that it can not be quantitated in any reliable manner. While increasing the concentration of the decalcification agent has been found to speed decalcification, it also increases the rate of degradation of the RNA in the sample.
Accordingly, there is a need in the art for a method for the rapid decalcification of hard tissues like bone, mineralizing cartilage and tendon, dentin, cementum and other tissues that are too hard to section effectively using conventional means, which preserves the ability to determine the site of gene expression without significantly degrading the RNA recovered for analysis.